PROJECT SUMMARY There is a clear unmet need for effective treatments for acute myeloid leukemia (AML). An exciting new mechanism for targeting cancer cells involves the activation of the extrinsic apoptotic pathway by administering the soluble domain of TNF-related apoptosis-inducing ligand (TRAIL), called dulanermin. Much hope has been placed on this drug since TRAIL has been shown to bind to death receptors and activate the extrinsic apoptotic pathway in cancer cells while having little impact on normal tissue. Dulanermin has completed several clinical trials, and while showing little toxicity, it has also disappointingly shown little efficacy. The main reasons attributed to dulanermin?s failure have been its low in vivo stability and several resistance mechanisms. To counter these issues, we propose a molecule that is composed of three tandem TRAIL domains (TR3) linked to an engineered safer version of L-asparaginase (called Asp) that we previously reported. For dulanermin to activate the death receptors, it must assemble into a trimer. Unlike monomeric dulanermin, TR3, by virtue of having the 3 TRAIL domains on a single polypeptide, results in a stabilized trimer that can better engage the death receptors. In addition to binding, clustering of the death receptors is also a key factor required for their activation. Asp is a tetrameric enzyme, so the linking of TR3 to Asp, which we refer to as TR3-Asp, results in a molecule that assembles into a tetramer with Asp at its core and 4 TRAIL trimers extending outside of it. In addition to increasing the clustering power of the biologic, the depletion of asparagine by the Asp reduces the amount of key TRAIL resistance factors such as cFLIP, XIAP and simultaneously increases the expression of death receptor 5. Additionally, several AML subtypes are inherently sensitive to asparagine depletion, so the addition of Asp to TRAIL also serves to directly target those L-asparaginase sensitive AML subtypes. Of particular note, by engineering out the toxicity-related L-glutaminase activity, our improved safer version ? Asp is very much compatible with the proven safety profile of TRAIL. Our strong preliminary data demonstrated that TR3-Asp has exceptional AML cell-killing power both in vitro and in mice. Notably, the mice under prolonged drug treatment exhibited no clinical signs of toxicity or weight loss. To build upon these exciting results we propose to further develop and evaluate TR3-Asp. Specifically, in Aim 1 we will optimize the production of TR3- Asp and use the identified conditions to manufacture the drug for the biological studies. In Aim 2 we will evaluate the pharmacokinetic properties and maximum tolerable dose of TR3-Asp. These data will inform the dosing of the in vivo evaluation of the molecule in patient derived xenografts, which are accepted as superior models of the human disease compared to cell line xenografts. Success in this work would provide the rationale for further development of TR3-Asp as a novel AML therapeutic, as well as in other cancers - such as pancreatic cancer - that have shown sensitivity to the individual TRAIL and L-asparaginase components.